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1.
Chem Pharm Bull (Tokyo) ; 68(1): 46-57, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31902901

RESUMO

Over the past decade, a number of new 1,4-naphthoquinones have been isolated from natural sources and new 1,4-naphthoquinones with diverse structural features have been synthesized. Cardioprotective, anti-ischemic, hepatoprotective, neuroprotective and some other new properties were found for these compounds; their role in protecting against neurodegenerative diseases has been established. Their anti-inflammatory, antimicrobial and antitumor activities have been studied in more detail; new, previously unknown intracellular molecular targets and mechanisms of action have been discovered. Some compounds of this class are already being used as a medicinal drugs and some substances can be used as biochemical tools and probes for non-invasive detection of pathological areas in cells and tissues in myocardial infarction and neurodegenerative diseases using modern molecular imaging techniques.


Assuntos
Anti-Infecciosos/química , Anti-Inflamatórios/química , Naftoquinonas/química , Substâncias Protetoras/química , Anti-Infecciosos/farmacologia , Anti-Inflamatórios/farmacologia , Bactérias/efeitos dos fármacos , Citocinas/metabolismo , Humanos , Leucócitos Mononucleares/citologia , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/metabolismo , Naftoquinonas/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Substâncias Protetoras/farmacologia , Trypanosoma/efeitos dos fármacos
2.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi ; 35(10): 872-877, 2019 Oct.
Artigo em Chinês | MEDLINE | ID: mdl-31814562

RESUMO

Objective To detect the effect of ethanol on T lymphocyte subsets, immune activity and apoptosis in peripheral blood. Methods Peripheral blood mononuclear cells (PBMCs) were isolated from healthy donors by density centrifugation. Lymphocytes were co-cultured with phytohaemagglutinin (PHA) in 30 and 50 mL/L ethanol. At 12, 18, 36, 48 hours after PBMCs were exposed to alcohol, flow cytometry was performed to examine the alterations of T lymphocyte subsets. Results Compared with the control group, at 12 hours after the exposure, the number of CD8+CD69+ T lymphocytes increased significantly in both PHA and alcohol groups but the number of CD8+CD69+ T lymphocytes in the PHA group was apparently higher than that of 50 ml/L ethanol group, while the number of T cells had no obvious change. At 18 hours after exposed to ethanol, the proliferation of T lymphocytes was reduced obviously in the ethanol groups. In addition, the proliferation of T cells in the ethanol (50 ml/L) group was markedly lower than that in 30 ml/L ethanol group. At the same time, the number of CD8+CD69+ T cells was markedly lower. At 36 hours after exposed to ethanol, in comparison with the PHA group, the number of T lymphocytes decreased markedly in the ethanol groups. At 48 hours after exposed to ethanol, T cells almost disappeared in the ethanol groups. The apoptosis rate of CD3+ T lymphocytes increased with the extension of co-culture time of PBMCs and ethanol as well as the increase of ethanol dose. Conclusion With the increase of ethanol dose, the activity of T lymphocytes in the peripheral blood treated with higher dose of ethanol would go down and the number of T lymphocyte subsets and the function of T cells decrease, which might be related to the ethanol that promotes the apoptosis of T lymphocyte subsets.


Assuntos
Apoptose , Etanol/farmacologia , Ativação Linfocitária , Subpopulações de Linfócitos T/efeitos dos fármacos , Células Cultivadas , Citometria de Fluxo , Humanos , Leucócitos Mononucleares/citologia , Subpopulações de Linfócitos T/citologia
3.
Photochem Photobiol Sci ; 18(12): 3008-3015, 2019 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-31696896

RESUMO

A quinoline moiety was used as a building block for designing a probe for the selective detection of copper ions in a partially aqueous medium. We have developed a molecular sensing system which gives insight into the complex physiological and redox aspects of labile copper. The probe provides a colorimetric approach for distinguishing cuprous and cupric ions along with their simultaneous discrimination from other metal ions in the visible range of the spectrum. The chemosensor showed a remarkable fluorescence enhancement along with a significant bathochromic shift of about 35 nm. The detection limit of the probe was found to be 1.03 µM which is optimally favorable to be applied in real-time monitoring. Fabrication of paper strips with the probe was done to detect the presence of cuprous ions in the real sample. The value of the binding constant (1.37 × 104 M-1) suggests stable complex formation between the metal ion and the sensing probe. The photoluminescence and structural aspects of the chemosensor were characterized by using fluorescence, absorption, ESI-MS, and 1H NMR spectroscopy. Furthermore, the cytotoxic nature and bioimaging properties of the probe were interpreted in vitro on RAW 264.7 macrophage cell lines and peripheral blood mononuclear cells (PBMCs) respectively.


Assuntos
Colorimetria , Cobre/análise , Quinolinas/química , Animais , Corantes Fluorescentes/química , Humanos , Íons/química , Leucócitos Mononucleares/química , Leucócitos Mononucleares/citologia , Limite de Detecção , Macrófagos/química , Macrófagos/citologia , Camundongos , Microscopia de Fluorescência , Células RAW 264.7
4.
Cell Physiol Biochem ; 53(5): 774-793, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31647207

RESUMO

BACKGROUND/AIMS: Deregulation of the complex interaction among host genetics, gut microbiota and environmental factors on one hand and aberrant immune responses on the other hand, are known to be associated with the development of inflammatory bowel disease. Recent studies provided strong evidence that autophagy plays a key role in the etiology of Crohn's disease (CD). Probiotics may exhibit many therapeutic properties, including anti-inflammatory abilities. While successful results have been obtained in ulcerative colitis patients, probiotics remain inefficient in CD for unknown reason. It remains therefore important to better understand their molecular mechanisms of action. METHODS: The activation of autophagy was examined by stimulating bone marrow-derived dendritic cells by the bacteria, followed by confocal microscopy and western blot analysis. The impact of blocking in vitro autophagy was performed in peripheral blood mononuclear cells using 3-methyl adenine or bafilomycin followed by cytokine secretion measurement by ELISA. The role of autophagy in the anti-inflammatory capacities of the bacterial strains was evaluated in vivo using an acute trinitrobenzene sulfonic acid-induced murine model of colitis. The impact of BMDC was evaluated by adoptive transfer, notably using bone marrow cells derived from autophagy-related 16-like 1-deficient mice. RESULTS: We showed that selected lactobacilli and bifidobacteria are able to induce autophagy activation in BMDCs. Blocking in vitro autophagy abolished the capacity of the strains to induce the release of the anti-inflammatory cytokine interleukin-10, while it exacerbated the secretion of the pro-inflammatory cytokine interleukin-1ß. We confirmed in the TNBS-induced mouse model of colitis that autophagy is involved in the protective capacity of these selected strains, and showed that dendritic cells are involved in this process. CONCLUSION: We propose autophagy as a novel mechanism involved in the regulatory capacities of probiotics.


Assuntos
Autofagia , Bifidobacterium/fisiologia , Lactobacillus/fisiologia , Adenina/análogos & derivados , Adenina/farmacologia , Animais , Proteínas Relacionadas à Autofagia , Células da Medula Óssea/citologia , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Quimiocinas/genética , Quimiocinas/metabolismo , Colite/induzido quimicamente , Colite/microbiologia , Colite/patologia , Citocinas/genética , Citocinas/metabolismo , Células Dendríticas/citologia , Células Dendríticas/metabolismo , Células Dendríticas/microbiologia , Feminino , Humanos , Leucócitos Mononucleares/citologia , Leucócitos Mononucleares/metabolismo , Macrolídeos/farmacologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout
5.
Exp Parasitol ; 206: 107755, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31493393

RESUMO

The aim of the present study was to assess the expression of cytokines and FCεR1A receptor stimulated by Haemonchus placei larval excretory and secretory (ES) products associated with the pathogenesis in calves. Bovine peripheral blood mononuclear cells (PBMC) were stimulated in in vitro assays with H. placei L4 ES product at 8, 12, 16 and 24 h. ES products were collected in in vitro assays at 48 h with molecular weight of 72/60 kDa and isoelectric point of 7.2 pI. Specific IgG for infected and control calves, positive and negative, were employed to recognise H. placei larval ES products by indirect ELISA, showing a mean of 1.8, 0.83 and 0.28 OD, respectively, (p ≤ 0.001). The quantification of relative gene expression was performed using a set of cytokines (IL-2, IFNγ, TGFß, IL-4, IL-5, IL-6, IL-8, IL-10 and IL-13), FCεR1A receptor and housekeeping (GAPDH, ß-actin and ß-2-microglobulin) by RT-qPCR. An early increased expression, 2.2- to 3.4-fold change, of IL-2 (p ≤ 0.001), IL-5 and TGFß (p ≥ 0.05) was determined, followed by TGFß (30.7 and 14.14), IL-8 (102.8 and 1504.4) and IL-10 (60.4 and 1.7) (p ≤ 0.05) after 12 and 16 h, respectively, and reducing the expression level at 24 h. In addition, IL-6, IL-13 and FCεR1A receptor also displayed mild expression level, 2.1 - to 7.60-fold change, at 24 h (p ≥ 0.05). We conclude that ES products of 72/60 kDa collected in vitro from H. placei larvae are recognised by infected hosts and have the ability to induce diverse immune factors to modulate the nematode damage.


Assuntos
Citocinas/metabolismo , Haemonchus/metabolismo , Leucócitos Mononucleares/citologia , Leucócitos Mononucleares/imunologia , Receptores de IgE/metabolismo , Animais , Bovinos , Proliferação de Células/efeitos dos fármacos , Citocinas/genética , Haemonchus/genética , Haemonchus/imunologia , Imunoglobulina G/metabolismo , Larva/genética , Larva/imunologia , Larva/metabolismo , Leucócitos Mononucleares/metabolismo , Masculino , RNA Mensageiro/metabolismo , Regulação para Cima
6.
Nat Methods ; 16(8): 715-721, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31363220

RESUMO

Accurately modeling cellular response to perturbations is a central goal of computational biology. While such modeling has been based on statistical, mechanistic and machine learning models in specific settings, no generalization of predictions to phenomena absent from training data (out-of-sample) has yet been demonstrated. Here, we present scGen (https://github.com/theislab/scgen), a model combining variational autoencoders and latent space vector arithmetics for high-dimensional single-cell gene expression data. We show that scGen accurately models perturbation and infection response of cells across cell types, studies and species. In particular, we demonstrate that scGen learns cell-type and species-specific responses implying that it captures features that distinguish responding from non-responding genes and cells. With the upcoming availability of large-scale atlases of organs in a healthy state, we envision scGen to become a tool for experimental design through in silico screening of perturbation response in the context of disease and drug treatment.


Assuntos
Algoritmos , Biologia Computacional/métodos , Leucócitos Mononucleares/metabolismo , Aprendizado de Máquina , Fagócitos/metabolismo , Análise de Célula Única/métodos , Transcriptoma , Animais , Simulação por Computador , Perfilação da Expressão Gênica , Humanos , Leucócitos Mononucleares/citologia , Camundongos , Fagócitos/citologia , Especificidade da Espécie
7.
Nucleic Acids Res ; 47(15): e89, 2019 09 05.
Artigo em Inglês | MEDLINE | ID: mdl-31165870

RESUMO

Optical DNA mapping (ODM) allows visualization of long-range sequence information along single DNA molecules. The data can for example be used for detecting long range structural variations, for aiding DNA sequence assembly of complex genomes and for mapping epigenetic marks and DNA damage across the genome. ODM traditionally utilizes sequence specific marks based on nicking enzymes, combined with a DNA stain, YOYO-1, for detection of the DNA contour. Here we use a competitive binding approach, based on YOYO-1 and netropsin, which highlights the contour of the DNA molecules, while simultaneously creating a continuous sequence specific pattern, based on the AT/GC variation along the detected molecule. We demonstrate and validate competitive-binding-based ODM using bacterial artificial chromosomes (BACs) derived from the human genome and then turn to DNA extracted from white blood cells. We generalize our findings with in-silico simulations that show that we can map a vast majority of the human genome. Finally, we demonstrate the possibility of combining competitive binding with enzymatic labeling by mapping DNA damage sites induced by the cytotoxic drug etoposide to the human genome. Overall, we demonstrate that competitive-binding-based ODM has the potential to be used both as a standalone assay for studies of the human genome, as well as in combination with enzymatic approaches, some of which are already commercialized.


Assuntos
Benzoxazóis/química , Mapeamento Cromossômico/métodos , DNA/química , Genoma Humano , Netropsina/química , Compostos de Quinolínio/química , Análise de Sequência de DNA/métodos , Antineoplásicos Fitogênicos/farmacologia , Sítios de Ligação , Ligação Competitiva , Cromossomos Artificiais Bacterianos/química , DNA/genética , Etoposídeo/farmacologia , Corantes Fluorescentes/química , Humanos , Leucócitos Mononucleares/citologia , Leucócitos Mononucleares/metabolismo , Imagem Individual de Molécula/métodos
9.
Cells ; 8(6)2019 06 11.
Artigo em Inglês | MEDLINE | ID: mdl-31212633

RESUMO

The human leukocyte antigen HLA-B27 is a strong risk factor for Ankylosing Spondylitis (AS), an immune-mediated disorder affecting axial skeleton and sacroiliac joints. Additionally, evidence exists sustaining a strong protective role for HLA-B27 in viral infections. These two aspects could stem from common molecular mechanisms. Recently, we have found that the HLA-B*2705 presents an EBV epitope (pEBNA3A-RPPIFIRRL), lacking the canonical B27 binding motif but known as immunodominant in the HLA-B7 context of presentation. Notably, 69% of B*2705 carriers, mostly patients with AS, possess B*2705-restricted, pEBNA3A-specific CD8+ T cells. Contrarily, the non-AS-associated B*2709 allele, distinguished from the B*2705 by the single His116Asp polymorphism, is unable to display this peptide and, accordingly, B*2709 healthy subjects do not unleash specific T cell responses. Herein, we investigated whether the reactivity towards pEBNA3A could be a side effect of the recognition of the natural longer peptide (pKEBNA3A) having the classical B27 consensus (KRPPIFIRRL). The stimulation of PBMC from B*2705 positive patients with AS in parallel with both pEBNA3A and pKEBNA3A did not allow to reach an unambiguous conclusion since the differences in the magnitude of the response measured as percentage of IFNγ-producing CD8+ T cells were not statistically significant. Interestingly, computational analysis suggested a structural shift of pEBNA3A as well as of pKEBNA3A into the B27 grooves, leaving the A pocket partially unfilled. To our knowledge this is the first report of a viral peptide: HLA-B27 complex recognized by TCRs in spite of a partially empty groove. This implies a rethinking of the actual B27 immunopeptidome crucial for viral immune-surveillance and autoimmunity.


Assuntos
Linfócitos T CD8-Positivos/metabolismo , Epitopos/imunologia , Antígeno HLA-B27/genética , Herpesvirus Humano 4/metabolismo , Espondilite Anquilosante/diagnóstico , Alelos , Sequência de Aminoácidos , Linfócitos T CD8-Positivos/citologia , Linfócitos T CD8-Positivos/imunologia , Epitopos/química , Humanos , Interferon gama/metabolismo , Leucócitos Mononucleares/citologia , Leucócitos Mononucleares/metabolismo , Ativação Linfocitária , Plasmídeos/genética , Plasmídeos/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Espondilite Anquilosante/imunologia , Espondilite Anquilosante/metabolismo , Espondilite Anquilosante/patologia
10.
J Vet Med Sci ; 81(6): 808-816, 2019 Jun 06.
Artigo em Inglês | MEDLINE | ID: mdl-31061249

RESUMO

We investigated the relationships between ruminal pH, gene expression in the rumen epithelium (RE), peripheral blood mononuclear cell subpopulations, and blood metabolites in Holstein calves during weaning transition. Calves (Weaning group, n=7) were assigned to one of two groups, and fed calf starter with forage (Forage group, n=3) or without forage (Starter group, n=4). Ruminal pH was measured continuously. Samples were collected at -1, 0, 1, and 3 weeks (blood and rumen fluid) or 3 weeks (rumen epithelium) after weaning. In the Weaning group, ruminal pH increased, and several blood metabolites increased (blood urea nitrogen [BUN], beta-hydroxybutyrate [BHB], and gamma-glutamyl transferase [GGT]) or decreased (total cholesterol [T-CHO] and phospholipid) after weaning. Ruminal pH was positively correlated with CD8+CD45R- cell populations and blood metabolites (BUN, glucose, and BHB) and negatively correlated with GGT activity. The 24 hr mean ruminal pH was higher in the Forage group during weaning transition, and toll-like receptor 4 mediated signaling pathway was activated in the Starter group at 3 weeks post-weaning. The number of CD8+CD45R- cells tended to be higher, and several blood metabolites (glucose, triglycerides, T-CHO, and phospholipid) were higher in the Forage group after weaning. Calves with higher ruminal pH also showed a greater energy metabolism status simultaneously with lesser hepatic disturbance enzymes in the peripheral blood. The results of our study indicate that serum GGT activity may be a plausible biomarker for predicting ruminal acidosis in Holstein calves during weaning transition.


Assuntos
Fenômenos Fisiológicos da Nutrição Animal , Bovinos/metabolismo , Dieta/veterinária , Rúmen/química , Desmame , Ração Animal/análise , Animais , Biomarcadores/sangue , Epitélio/metabolismo , Regulação da Expressão Gênica , Concentração de Íons de Hidrogênio , Leucócitos Mononucleares/citologia , Masculino , Rúmen/metabolismo , Transdução de Sinais , Receptor 4 Toll-Like , gama-Glutamiltransferase/sangue
11.
J Diabetes Res ; 2019: 5184647, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31143779

RESUMO

Background: The syndrome of maternally inherited diabetes and deafness (MIDD) is typically caused by the m.3243A>G mutation and widely considered maternally inherited. In our study, we aimed to investigate the heredity way of the m.3243A>G among pedigrees with MIDD and discover novel mitochondrial DNA mutations related to atypical clinical phenotypes. Methods: Heteroplasmy levels of the m.3243A>G mutation in peripheral blood, saliva, and urine sediment of 31 individuals from 10 unrelated pedigrees were measured by pyrosequencing. Clinical evaluations including endocrinological, audiological, and magnetic resonance imaging (MRI) examinations, mitochondrial function evaluation in peripheral blood mononuclear cells (PBMCs), and whole mitochondrial DNA (mtDNA) sequencing were performed among the spontaneous mutant pedigrees. Results: Among the 10 unrelated MIDD pedigrees, we found that the de novo m.3243A>G mutation occurred in the family 1957 (F1957). The proband (F1957-II-1) and her son (F1957-III-1) both manifested diabetes with mild bilateral sensorineural hearing loss (SNHL) and abnormal brain MRI, and F1957-III-1 also complained of severe nausea and vomiting. Mitochondrial function evaluation in PBMCs revealed an increased level of ROS generation and decreased levels of ATP and mitochondrial membrane potential (ΔΨm) in the two m.3243A>G carriers. Whole mtDNA sequencing also revealed a de novo heteroplasmic substitution at m.16093T>C in both the proband and her son. Conclusions: Our study showed that de novo m.3243A>G mutation accompanied by other point mutations may occur in the very early embryonic or germ cell stage without maternal inheritance, bringing about both typical and atypical clinical features.


Assuntos
DNA Mitocondrial/genética , Surdez/genética , Surdez/fisiopatologia , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/fisiopatologia , Leucina/genética , Doenças Mitocondriais/genética , Doenças Mitocondriais/fisiopatologia , RNA de Transferência/genética , Trifosfato de Adenosina/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Saúde da Família , Feminino , Predisposição Genética para Doença , Humanos , Leucócitos Mononucleares/citologia , Imagem por Ressonância Magnética , Masculino , Pessoa de Meia-Idade , Mutação , Linhagem , Fenótipo , Mutação Puntual , Espécies Reativas de Oxigênio/metabolismo , Análise de Sequência de DNA
12.
J Nanobiotechnology ; 17(1): 72, 2019 May 27.
Artigo em Inglês | MEDLINE | ID: mdl-31133024

RESUMO

BACKGROUND: Nano-sized vesicles, so called extracellular vesicles (EVs), from regenerative cardiac cells represent a promising new therapeutic approach to treat cardiovascular diseases. However, it is not yet sufficiently understood how cardiac-derived EVs facilitate their protective effects. Therefore, we investigated the immune modulating capabilities of EVs from human cardiac-derived adherent proliferating (CardAP) cells, which are a unique cell type with proven cardioprotective features. RESULTS: Differential centrifugation was used to isolate EVs from conditioned medium of unstimulated or cytokine-stimulated (IFNγ, TNFα, IL-1ß) CardAP cells. The derived EVs exhibited typical EV-enriched proteins, such as tetraspanins, and diameters mostly of exosomes (< 100 nm). The cytokine stimulation caused CardAP cells to release smaller EVs with a lower integrin ß1 surface expression, while the concentration between both CardAP-EV variants was unaffected. An exposure of either CardAP-EV variant to unstimulated human peripheral blood mononuclear cells (PBMCs) did not induce any T cell proliferation, which indicates a general low immunogenicity. In order to evaluate immune modulating properties, PBMC cultures were stimulated with either Phytohemagglutin or anti-CD3. The treatment of those PBMC cultures with either CardAP-EV variant led to a significant reduction of T cell proliferation, pro-inflammatory cytokine release (IFNγ, TNFα) and increased levels of active TGFß. Further investigations identified CD14+ cells as major recipient cell subset of CardAP-EVs. This interaction caused a significant lower surface expression of HLA-DR, CD86, and increased expression levels of CD206 and PD-L1. Additionally, EV-primed CD14+ cells released significantly more IL-1RA. Notably, CardAP-EVs failed to modulate anti-CD3 triggered T cell proliferation and pro-inflammatory cytokine release in monocultures of purified CD3+ T cells. Subsequently, the immunosuppressive feature of CardAP-EVs was restored when anti-CD3 stimulated purified CD3+ T cells were co-cultured with EV-primed CD14+ cells. Beside attenuated T cell proliferation, those cultures also exhibited a significant increased proportion of regulatory T cells. CONCLUSIONS: CardAP-EVs have useful characteristics that could contribute to enhanced regeneration in damaged cardiac tissue by limiting unwanted inflammatory processes. It was shown that the priming of CD14+ immune cells by CardAP-EVs towards a regulatory type is an essential step to attenuate significantly T cell proliferation and pro-inflammatory cytokine release in vitro.


Assuntos
Doenças Cardiovasculares/terapia , Vesículas Extracelulares/imunologia , Monócitos/imunologia , Miócitos Cardíacos/imunologia , Doenças Cardiovasculares/imunologia , Linhagem Celular , Proliferação de Células , Técnicas de Cocultura , Citocinas/metabolismo , Vesículas Extracelulares/metabolismo , Humanos , Imunomodulação , Leucócitos Mononucleares/citologia , Leucócitos Mononucleares/imunologia , Monócitos/citologia , Miócitos Cardíacos/citologia , Regeneração , Linfócitos T/citologia , Linfócitos T/imunologia
13.
Tohoku J Exp Med ; 248(1): 45-55, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-31130587

RESUMO

The Tohoku Medical Megabank biobank (TMM biobank) is the first major population-based biobank established in Japan. The TMM biobank was established based on two population cohorts and is a reconstruction program from the Great East Japan Earthquake and Tsunami of 2011. The biobank stores more than 3.4 million tubes of biospecimens and associated health and analytic data obtained from approximately 150,000 TMM cohort participants between May 2013 and December 2018, and the TMM biobank currently shares high-quality specimens and data. Various biospecimens, including peripheral and cord blood mononuclear cells, buffy coat, plasma, serum, urine, breast milk and saliva have been collected in the TMM biobank. To minimize human error and maintain the quality of data and specimens, we have been utilizing laboratory information management system into various biobank procedures from registration to storage with various automation systems, such as liquid dispensing, DNA extraction and their storage. The biobank procedures for the quality management system (ISO 9001:2015) and information security management system (ISO 27001:2013) are certified by the International Organization for Standardization. The quality of our biobank samples fulfills the pre-analytical requirements for researchers conducting next-generation whole genome sequencing, DNA array analyses, proteomics, metabolomics, etc. We established analytical centers to conduct standard genomic and multiomic analyses in-house and share the generated data. Additionally, we generate thousands of Epstein-Barr virus (EBV)-transformed lymphoblastoid cell lines and proliferating T cells for functional studies. The TMM biobank serves as an indispensable infrastructure for academic, clinical and industrial research to actualize next-generation medicine in Japan.


Assuntos
Bancos de Espécimes Biológicos , Manejo de Espécimes , Bancos de Espécimes Biológicos/normas , Estudos de Coortes , DNA/isolamento & purificação , Humanos , Disseminação de Informação , Japão , Leucócitos Mononucleares/citologia , Controle de Qualidade , Transportes
14.
Int J Mol Sci ; 20(10)2019 May 26.
Artigo em Inglês | MEDLINE | ID: mdl-31130703

RESUMO

Co-culture studies investigating the role of periprosthetic fibroblasts (PPFs) in inflammatory osteoclastogenesis reveal contrary results, partly showing an osteoprotective function of fibroblasts and high OPG expression in monolayer. These data disagree with molecular analyses of original periosteolytic tissues. In order to find a more reliable model, PPFs were co-cultivated with peripheral blood mononuclear cells (PBMCs) in a transwell system and compared to conventional monolayer cultures. The gene expression of key regulators of osteoclastogenesis (macrophage colony-stimulating factor (MCSF), receptor activator of NF-κB ligand (RANK-L), osteoprotegerin (OPG), and tumor necrosis factor alpha (TNFα)) as well as the ability of bone resorption were analyzed. In monolayer co-cultures, PPFs executed an osteoprotective function with high OPG-expression, low RANK-L/OPG ratios, and a resulting inhibition of osteolysis even in the presence of MCSF and RANK-L. For transwell co-cultures, profound changes in gene expression, with a more than hundredfold decrease of OPG and a significant upregulation of TNFα were observed. In conclusion, we were able to show that a change of culture conditions towards a transwell system resulted in a considerably more osteoclastogenic gene expression profile, being closer to findings in original periosteolytic tissues. This study therefore presents an interesting approach for a more reliable in vitro model to examine the role of fibroblasts in periprosthetic osteoclastogenesis in the future.


Assuntos
Fibroblastos/citologia , Leucócitos Mononucleares/citologia , Osteoclastos/citologia , Osteogênese , Idoso , Células Cultivadas , Técnicas de Cocultura/métodos , Feminino , Humanos , Masculino , Pessoa de Meia-Idade
15.
Blood Coagul Fibrinolysis ; 30(4): 133-139, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31090595

RESUMO

: An increased T-helper cell (Th) 1/Th2 ratio in the peripheral blood has been proposed to correlate with the disease activity of immune thrombocytopenia (ITP). T-cell immunoglobulin and mucin domain-containing molecule 3 (TIM-3) is a Th1-associated cell surface molecule that regulates Th1 responses and promotes tolerance. Consequently, we aimed to determine whether the regulation of TIM-3 expression is likely to be a promising therapeutic approach for ITP. In the present study, we investigated the immunomodulatory activities of TIM-3 in human peripheral blood mononuclear cell (PBMC) cultures. Levels of interferon-gamma (IFN-γ), tumor necrosis factor alpha (TNF-α), interleukin (IL)-4, IL-5, IL-2, and IL-10 were determined in PBMCs from 11 ITP patients and 10 healthy patients after TIM-3 antibody administration for 48 h. The proliferation of PBMCs was examined by cell counting kit-8 assay. Flow cytometry was used to observe apoptosis by staining cells with annexin V-fluorescein isothiocyanate/propidine iodide. PBMCs from ITP patients secreted higher amounts of IFN-γ than those from control patients but paradoxically expressed lower levels of TIM-3. Depletion of TIM-3 in PBMCs in vitro using a TIM-3 antibody enhanced IFN-γ secretion, directly demonstrating that TIM-3 expression on human T cells regulates proliferation and IFN-γ secretion. Failure to upregulate the T-cell expression of TIM-3 may represent a novel intrinsic defect that contributes to the pathogenesis of ITP.


Assuntos
Receptor Celular 2 do Vírus da Hepatite A/metabolismo , Púrpura Trombocitopênica Idiopática/terapia , Células Th1/imunologia , Apoptose , Estudos de Casos e Controles , Proliferação de Células , Células Cultivadas , Citocinas/metabolismo , Regulação da Expressão Gênica , Receptor Celular 2 do Vírus da Hepatite A/antagonistas & inibidores , Humanos , Leucócitos Mononucleares/citologia , Púrpura Trombocitopênica Idiopática/etiologia , Púrpura Trombocitopênica Idiopática/metabolismo , Púrpura Trombocitopênica Idiopática/patologia , Células Th1/metabolismo
16.
Genes (Basel) ; 10(4)2019 04 12.
Artigo em Inglês | MEDLINE | ID: mdl-31013806

RESUMO

Infection with canine heartworm (Dirofilaria immitis), spread via mosquito vectors, causes coughing, asthma, pneumonia, and bronchitis in humans and other animals. The disease is especially severe and often fatal in dogs and represents a serious threat to public health worldwide. Cysteine protease inhibitors (CPIs), also known as cystatins, are major immunomodulators of the host immune response during nematode infections. Herein, we cloned and expressed the cystatin Di-CPI from D. immitis. Sequence analysis revealed two specific cystatin-like domains, a Q-x-V-x-G motif, and a SND motif. Phylogenetic analysis indicates that Di-CPI is a member of the second subgroup of nematode type II cystatins. Probing of D. immitis total proteins with anti-rDi-CPI polyclonal antibody revealed a weak signal, and immunofluorescence-based histochemical analysis showed that native Di-CPI is mainly localized in the cuticle of male and female worms and the gut of male worms. Treatment of canine peripheral blood mononuclear cells (PMBCs) with recombinant Di-CPI induced a Th2-type immune response characterized by high expression of the anti-inflammatory factor interleukin-10. Proliferation assays showed that Di-CPI inhibits the proliferation of canine PMBCs by 15%. Together, the results indicate that Di-CPI might be related to cellular hyporesponsiveness in dirofilariasis and may help D. immitis to evade the host immune system.


Assuntos
Clonagem Molecular/efeitos dos fármacos , Cistatinas/genética , Cistatinas/metabolismo , Dirofilaria immitis/enzimologia , Análise de Sequência de DNA/métodos , Animais , Anticorpos/metabolismo , Células Cultivadas , Cistatinas/química , Cistatinas/imunologia , Dirofilaria immitis/genética , Dirofilaria immitis/imunologia , Cães , Feminino , Trato Gastrointestinal/metabolismo , Proteínas de Helminto/química , Proteínas de Helminto/genética , Proteínas de Helminto/imunologia , Proteínas de Helminto/metabolismo , Evasão da Resposta Imune , Interleucina-10/metabolismo , Leucócitos Mononucleares/citologia , Leucócitos Mononucleares/imunologia , Masculino , Filogenia , Domínios Proteicos , Coelhos , Distribuição Tecidual
17.
Immun Inflamm Dis ; 7(3): 105-111, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31016894

RESUMO

INTRODUCTION: The information content of multiparametric flow cytometry experiments is routinely underexploited given the paucity of adequate tools for unbiased comprehensive data analysis that can be applied successfully and independently by immunologists without computational training. METHODS: We aimed to develop a tool that allows straightforward access to the entire information content of any given flow cytometry panel for immunologists without special computational expertise. We used a data analysis approach which accounts for all mathematically possible combinations of markers in a given panel, coded the algorithm and applied the method to mined and self-generated data sets. RESULTS: We developed Flow Plex, a straightforward computational tool that allows unrestricted access to the information content of a given flow cytometry panel, enables classification of human samples according to distinct immune phenotypes, such as different forms of autoimmune uveitis, acute myeloid leukemia vs "healthy", "old" vs "young", and facilitates the identification of cell populations with potential biologic relevance to states of disease and health. CONCLUSIONS: We provide a tool that allows immunologists and other flow cytometry users with limited bioinformatics skills to extract comprehensive, unbiased information from flow cytometry data sets.


Assuntos
Biologia Computacional/métodos , Análise de Dados , Citometria de Fluxo/métodos , Imunofenotipagem/métodos , Leucemia Mieloide/patologia , Leucócitos Mononucleares/citologia , Doença Aguda , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Algoritmos , Células Cultivadas , Criança , Análise por Conglomerados , Feminino , Humanos , Leucemia Mieloide/classificação , Leucócitos Mononucleares/classificação , Masculino , Pessoa de Meia-Idade , Fenótipo
18.
Methods Mol Biol ; 1979: 9-21, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31028629

RESUMO

The starting material for all single-cell protocols is a cell suspension. The particular functions and spatial distribution of immune cells generally make them easy to isolate them from the tissues where they dwell. Here we describe tissue dissociation protocols that have been used to obtain human immune cells from lymphoid and nonlymphoid tissues to be then used as input to single-cell methods. We highlight the main factors that can influence the final quality of single-cell data, namely the stress signatures that can bias its interpretation.


Assuntos
Perfilação da Expressão Gênica/métodos , RNA/genética , Análise de Sequência de RNA/métodos , Análise de Célula Única/métodos , Manejo de Espécimes/métodos , Fracionamento Celular/métodos , Humanos , Leucócitos Mononucleares/citologia , Leucócitos Mononucleares/metabolismo , Pulmão/citologia , Pulmão/metabolismo , Linfonodos/citologia , Linfonodos/metabolismo , Neutrófilos/citologia , Neutrófilos/metabolismo , RNA/sangue , RNA/isolamento & purificação , Pele/citologia , Pele/metabolismo , Baço/citologia , Baço/metabolismo
19.
Methods Mol Biol ; 1979: 155-176, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31028637

RESUMO

Peripheral blood mononuclear cells (PBMCs) are blood cells that are a critical part of the immune system used to fight off infection. However, due to the complexity of PBMCs, which contain multiple different cell types, studying the function of the individual cell types can be difficult, and often studies rely on bulk measurements. Here, we describe the analysis of PBMCs using single-cell RNA-sequencing in droplets. Data from these studies allow for the identification and quantification of the subpopulation of cells that make up the PBMC sample. In addition, differential gene expression between cell types and samples can be assessed.


Assuntos
Leucócitos Mononucleares/metabolismo , Análise de Sequência de RNA/métodos , Análise de Célula Única/métodos , Contagem de Células/métodos , Separação Celular/métodos , DNA Complementar/genética , Desenho de Equipamento , Sequenciamento de Nucleotídeos em Larga Escala/instrumentação , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Leucócitos Mononucleares/citologia , Receptores de Lipopolissacarídeos/análise , Monócitos/citologia , Monócitos/metabolismo , Transcrição Reversa , Análise de Sequência de RNA/instrumentação , Análise de Célula Única/instrumentação , Fluxo de Trabalho
20.
Methods Mol Biol ; 1979: 285-303, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31028645

RESUMO

Mass cytometry is a variation of conventional flow cytometry using metal tagged antibodies for cell staining instead of fluorochromes and detection in a mass cytometer, a modified mass spectrometer that allows for separation of discrete masses of these metal tags by time of flight (TOF). Currently, up to 50 different metal tags are available for cell analysis. The lack of any significant mass spectral overlap and autofluorescence background makes mass cytometry uniquely suited for complex high-dimensional phenotypic and functional analysis at the single cell level, thus accelerating biomarker discovery and drug screening. Here we describe a workflow for phenotyping of human peripheral blood mononuclear cells (PBMCs) covering cell staining, instrument setup of a Fluidigm Helios™ mass cytometer, and sample acquisition, and summarize a basic workflow of data analysis.


Assuntos
Citometria de Fluxo/métodos , Imunoconjugados/imunologia , Imunofenotipagem/métodos , Leucócitos Mononucleares/imunologia , Separação Celular/métodos , Sobrevivência Celular , Humanos , Imunoconjugados/química , Irídio/química , Irídio/imunologia , Isótopos/química , Isótopos/imunologia , Leucócitos Mononucleares/citologia , Ródio/química , Ródio/imunologia , Análise de Célula Única/métodos , Coloração e Rotulagem/métodos
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